1. Field of the Invention
The invention relates generally to the field of medical treatment in relation to assays such as those designed to assist in evaluating drug regimens. The invention also provides novel vectors useful for single cell analysis.
2. Description of the Background Art
Human Immunodeficiency Virus (HIV) is the causative agent for Acquired Immuno-Deficiency Syndrome (AIDS), one of the world's most feared diseases. HIV has been the cause of death for over 15 million people and millions more are infected with the HIV virus. It is estimated that in sub-Saharan Africa there are approximately 28 million people infected with HIV. The AIDS virus is unique in that it is insidious, infecting CD4 cells in the body. These cells are a type of white blood cell the body uses to fight infection, but after infection the CD4 cells produce virus, not more CD4 cells.
Once the CD4+ cell count has been reduced to less than 200, the immune defense has become seriously compromised, leaving the body vulnerable to opportunistic infections such as pneumonia, tuberculosis and some forms of cancer. In fact the cause of death in AIDS patients is usually from an infection and not directly from HIV.
There is no known cure for AIDS, nor has a protective vaccine been developed. There are medications that will slow viral replication and in many cases enable HIV-positive patients to live a near normal life. Treatments are expensive and have included use of antiretrovirals such as reverse transcriptase (RT) protease inhibitors (PIs). Current treatment of choice is use of combinations of drugs, each of which acts somewhat differently, with close monitoring of patient health so that dosages and combinations can be adjusted.
HAART
HIV treatment has evolved considerably since the first retroviral inhibitors were used. Current therapies employ a panel of therapeutics, known as Highly Active Anti-Retroviral Therapy (HAART). HAART is an aggressive therapy utilizing a multiple of anti-HIV drugs, not only for patients with AIDS but also to some HIV-positive subjects even before they develop symptoms of AIDS. The therapy commonly includes one nucleoside analog (such as a DNA chain terminator), one protease inhibitor and either a second nucleoside analog or a non-nucleoside reverse transcription inhibitor (NNRTI).
There are a number of drugs used in HAART therapy, of which azidothymidine (AZT) is one of the earliest and best-known nucleoside analogs used in treating AIDS. Other drugs used in combinational HAART therapies include zalcitabine (ddC), didanosine (ddI), amprenavir (AVP), Ritonavir (RTV), abacavir (ABC), tenofovir disoproxil fumarate, (TDF), nelfinavir (NFV), saquinavir (SQV), lopinavir (LPV) and indinavir (IDV)
Unfortunately, HAART is a difficult treatment because of toxicity, treatment failure or side effects Baron, et al. (2004). Adherence to prescribed regimens is not always easy to determine and patient compliance is often low, which may be due in part because there are no established standards for assessing compliance. The toxicity and tolerability of HAART are increasingly important factors in decisions relating to considering which of more than 6000 potential regimen combinations to prescribe or, perhaps more importantly, when to modify or discontinue a particular regimen.
Adverse effects are associated with each class of drug used in HAART. Nucleoside analogs have been reported to cause severe nucleoside associated lactic acidosis (NALA), or pancreatitis. The protease drugs are associated with hypertriglyceridemia, insulin resistance, diarrhea, nausea, headache and oral paresthesia. IL-2 combination therapies have been associated with fever, fatigue and myalgias.
Auxiliary therapy is often required in AIDS treatment because patients with compromised immune systems are susceptible to many types of infections, particularly pneumocystis carinii pneumonia (PCP) and tuberculosis. Adverse effects to drugs used in treatment of these conditions complicates AIDS therapy and may contribute to death of a patient.
Resistance to AIDS therapy is one of the most compelling problems in managing drug regimens. HIV RNA levels are the usual means of monitoring viral load and progress of the therapy.
Perhaps the most challenging aspect of the HIV is its ability to rapidly mutate in infected patients. In the United States at least, the infecting virus “wildtype” is HIV-1, although in some other parts of the world HIV-2 is more common. The number of naturally occurring mutations is high and many of the amino acid changes known to contribute to drug resistance occur as natural polymporphisms in isolates from patients who have never been treated with protease inhibitors (Kozal, et al. (1996). A great deal is known about the mutations that confer resistance to antiretroviral drugs, although development of drugs to effectively combat the mutant virus is relatively slow compared to the pace at which HIV mutations can appear.
Several assays have been used to monitor the development of drug resistance. Population-level sequencing of viruses in plasma can reveal the existence of characteristic mutations associated with drug resistance. Genotypic data can be used to predict drug resistance phenotypes by using compiled databases and established algorithms.
Direct phenotypic assays of drug resistance have also been developed (Petropoulos, et al, 2000) and are of particular value when multiple mutations are present. These assays use pooled HIV-1 reverse transcriptase (RT) and protease sequences amplified from plasma to measure susceptibility to individual antiretroviral drugs. The interpretation of these assays is complicated by the fact that viruses replicating in vivo experience simultaneous selection by each of the drugs in the regimen. The possible synergy and antagonism that may occur with treatment with multiple agents are not reflected in current assays. A particular problem is that current assays do not provide a clear indication of whether or not multiple antiretroviral drugs acting synergistically might still have some residual activity against viruses with resistance mutations. Thus, phenotypic assays that can compare the susceptibility of viral isolates to drug combinations, rather than to individual drugs, would be a valuable tool for choosing alternative regimens in the setting of treatment failure.
The choice of treatment regimens in the setting of failure is further complicated by the issue of replication capacity. Studies by Deeks et al. (2001) have demonstrated that some patients who are failing therapy maintain relatively high CD4 counts despite detectable viremia. Interruption of therapy leads to the loss of this immunologic benefit. Because some drug resistance mutations can reduce the fitness of the virus relative to wild-type virus in the absence of drugs (Nijhuis, et al. 2001), some investigators suggested that the immunologic benefit of continued treatment in the presence of virologic failure may reflect selection for drug-resistant mutants with diminished replication capacities (Barbour, et al., 2002). This benefit is entirely dependent upon the assumption that the wild-type virus with higher fitness is preserved and will reappear if therapy is stopped. Indeed, wild-type viruses do reappear several weeks after treatment interruption (Deeks, et al., 2001).
The reappearance of wild-type virus is unlikely to be simply genetic reversion because different forms of resistance involving either single mutations or accumulations of multiple mutations disappear with similar kinetics. Phylogenetic evidence suggests that the reemerging wild-type viruses are archival (Kijak, et al., 2002). At the present time, the only site in which wild-type viruses have been shown to persist despite prolonged replication of and selection for drug-resistant viruses is the latent reservoir in resting memory CD4+ T cells.
Deficiencies in the Art
A major concern in treating HIV patients receiving HAART is the ability of the virus to mutate, often resulting in less effective treatment or treatment failure. There is a need for methods that provide guidelines for determining whether or not to continue or adjust drug regimens for HIV patients. Patients failing HAART may nevertheless derive benefit from continued treatment for two reasons, namely the residual susceptibility of the resistant viruses to the drug regimen and the diminished replication capacities of the resistant viruses. Current assays do not provide a simple way to determine the relative importance of these two effects, making it difficult for the clinician to determine any potential benefit from continued therapy or rationally adjusted therapy with respect to the number and type of antiviral drugs administered.